Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States

ABSTRACT Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S. IMPORTANCE Aspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.


Sampling
Sampling was conducted as previously described (11).Briefly, between November 2018 and October 2019, soil, plant debris, or compost was collected from 29 agricultural sites in the eastern U.S. (New York, Pennsylvania, Virginia, South Carolina, and Georgia) and from 23 agricultural sites in the western U.S. (Washington, Oregon, and California; Table 1).Soil and compost were sampled by taking 3-5 soil cores to a depth of 10-15 cm.Fallen plant debris was collected with the soil if present.Collections were limited to four samples per site to minimize the isolation of clones.Samples were shipped overnight in sealed plastic bags and then stored at 4°C and with bags open to allow for gas exchange.Samples were generally processed within 2 weeks of collection.

Isolation and storage
The samples were processed as described previously with some modifications (11,18,20).Briefly, 2 g of soil was suspended in 8 mL of 0.1 M sterile sodium pyrophosphate.Samples were vortexed for 30 seconds and allowed to settle for 1 minute.From the supernatant, 100 µL was pipetted onto 10 plates each of Sabouraud dextrose agar (SDA) control plates or SDA supplemented with 3 µg/mL of the fungicide tebuconazole or with 4 µg/mL of the antifungal itraconazole.All plates additionally contained 50 µg/mL of chloramphenicol and 5 µg/mL of gentamicin to inhibit bacterial growth and 25 µg/mL of Rose-Bengal dye to reduce the growth of Rhizopus and Mucor.The plates were sealed with micropore tape and incubated at 45°C for 48-72 hours.Colonies of A. fumigatus were identified by morphology and the distinctive gray-green spore color.Colonies that grew on azole-drug amended media were single-colony isolation streaked on fresh azole-containing media without other antimicrobials added.Several colonies that grew on negative control plates were single-colony isolation streaked and plated to azoledrug amended and non-amended media to identify sensitive isolates for comparison in whole-genome sequencing (WGS) analyses.All collected isolates were stored in 15% glycerol at −80°C.

MIC assays for antifungal susceptibility testing
Using the Clinical Laboratory Standard Institute broth microdilution method for antifungal susceptibility testing, we tested 160 environmental A. fumigatus isolates for sensitivity to the fungicide tebuconazole (TEB; TCI America, Oregon, USA), and the antifungals itraconazole (ITC; Thermo Sci Acros Organics, New Jersey, USA), voriconazole (VOR; Thermo Sci Acros Organics, New Jersey, USA), and posaconazole (POS; Apexbio Technology, Texas, USA).The isolates were grown on complete media slants for 4 days and were then harvested using 3 mL of 0.05% Tween-20 to suspend the hydrophobic spores in solution.The spore suspensions were adjusted to an optical density of 0.09-0.13 at 530 nm using a spectrophotometer, and 100 µL of spore suspension was added to a microtiter plate well containing 100 µL of RPMI 1640 liquid medium (Thermo Sci Gibco, California, USA) and azoles with final concentrations ranging from 0 to 16 µg/mL.The plates were incubated for 48 hours at 37°C and then characterized by the European Committee on Antibiotic Susceptibility Testing (EUCAST) breakpoints (21).Minimum inhibitory concentration (MIC) (21) breakpoints were defined as the lowest concentration where 100% of growth was inhibited.The assay was performed twice on all 160 isolates.For instances where MIC break points differed by greater than one dilution between replicates, the isolates were assayed a third time.For classification of sensitivity or resistance for TEB (>3 µg/mL), ITC (> 2 µg/mL), VOR (> 2 µg/mL), and POS (> 0.25 µg/mL, if = to 0.25 µg/mL consider resistant if ITC is resistant), we used the current recommen ded clinical breakpoints of antifungal resistance for A. fumigatus published by EUCAST 2020 (21,22).

DNA extraction and sequencing
Genomic DNA was extracted from A. fumigatus isolates using a cetrimonium bromide (CTAB) protocol described previously with some modifications (11,23).Briefly, cultures were grown overnight in complete medium broth, and approximately 200 mg of mycelium was collected and transferred to 2 mL microcentrifuge tubes containing 150 mg of glass disruption beads and three 3 mm steel beads and lyophilized for 24 hours.Lyophilized cells were disrupted using GenoGrinder 2010 (OPS Diagnostics, Lebanon, NJ) at 1,750 rpm for 30 seconds.To the pulverized tissue, 1 mL of CTAB lysis buffer [100 mM Tris pH 8.0, 10 mM EDTA, 1% CTAB, and 1% β-mercaptoethanol (BME)] was added, and the samples were incubated for 30 minutes at 65°C.After incubation, 300 µL of 5M KAc was added to the samples and incubated on ice for 30 minutes.The supernatant was extracted twice with chloroform, and the aqueous, clear top layer containing DNA was precipitated by adding an equal volume of ice-cold 100% isopropa nol, followed by centrifugation.The DNA pellet was washed in 70% EtOH, then 100% EtOH, and air-dried.The resuspended sample was treated with RNase A according to the JGI RNase A DNA cleanup protocol.DNA was checked for quality and quantified using NanoDrop One (Thermo Sci, New Jersey, USA).Library preparation and Illumina NextSeq 2000 P3 sequencing were conducted at the Georgia Genomics and Bioinformatics Core at the University of Georgia, Athens, GA.

Variant calling and phylogenetic analysis
Raw reads were mapped to Af293 (GCF_000002655.1)using BWA v0.7.17 (29).Text pileup outputs were generated for each sequence using SAMtools v1.6 mpileup with option -I to exclude insertions and deletions (30).BCFtools v1.6 call was used to call single nucleotide polymorphisms (SNPs) with options -c to use the original calling method and --ploidy 1 for haploid data (31).Consensus genome sequences in fasta format were extracted from vcf files using seqtk v1.2 (available at https://github.com/lh3/seqtk) with bases with a phred quality score below 40 counted as missing data (N).Because insertions and deletions were removed when mapping reads to the reference, all genome sequences are already mapped to the same coordinates as the reference sequence, and there was, therefore, no need for a multiple alignment step.Variant files (vcf ) were used in vcf2allelePlot.plto test the B-Allele Frequency of all isolates to ensure they were derived from single spores of A. fumigatus (32).
MEGA X (33) was used to create a neighbor-joining tree using the Tamura-Nei model with 100 bootstraps for all whole-genome sequences.Neighbor joining is a suitable method for constructing whole-genome trees from SNP data within a frequently recombining species like A. fumigatus.Visualization was done using iTOL (34).MEGA X (33) was used to create neighbor-joining gene trees using the Kimura 2-parameter model (35) with 100 bootstraps for all housekeeping genes.

Population genetic analyses
Principal-component analysis (PCA) was performed using the smartpca program of EIGENSOFT v7.2.1 (36).PCA results were plotted using the ggplot2 package in R version 4.2.1 (37,38).All vcf files were merged using BCFtools v1.9 and thinned using VCFtools v0.1.16with options -thin to have one SNP for every 50 nucleotides, --minQ 40 to filter out low-quality SNPs, and -plink to output plink files (ped and map).Plink v1.9b was used with the ped and map files as input and option --make-bed to output a bed file to be used in ADMIXTURE (39).To identify ancestry and admixed strains, ADMIXTURE v 1.3 was used in five independent runs (1,12,345,33,333,54,321, and 98,765) with 2-20 clusters (K) (40).Resulting Q files were used in the Distruct for many K's features of CLUMPAK to align cluster labels across different models (41).CLUMPAK results were added to R version 3.5.0 to be plotted (37).Cross-validation values for all five runs K2-20 can be found in Fig. S4.The neighbor-joining tree, PCA, critical values (BIC), and loglikelihood values of each K were used to identify the most likely number of clusters (K).

RESULTS
To better understand the prevalence of azole-resistant A. fumigatus in U.S. agricultural environments, we collected soil, plant debris, and compost from 29 agricultural sites in five eastern states (New York, Pennsylvania, Virginia, South Carolina, and Georgia) and from 23 agricultural sites in three western states (Washington, Oregon, and California).Crops cultivated on our sites included grains, fruits, nuts, and ornamentals (Table 1).With the exception of Georgia, no surveys to detect azole-resistant A. fumigatus in agricultural settings have been reported for these eight states.We plated samples onto media containing either no azole, the agricultural fungicide TEB, or the clinical antifungal drug ITC and isolated a total of 727 isolates of A. fumigatus.Following this preliminary selection, we determined the MIC for the clinical antifungal agents ITC, VOR, and POS by broth dilution assays for 202 isolates representing all sites and including azole-sensitive and -resistant isolates.Of these isolates, 44 were sensitive to all three azoles tested, 24 were resistant to ITC, 25 were resistant to VOR, and 109 were resistant to POS, with 34 of those showing resistance to more than one clinical azole drug (pan-azole resistant) based on EUCAST 2020 cutoffs (Table 1) (21,22).We performed WGS on 135 isolates representing the range of collection sites and azole sensitivities.
Previous studies reported on clinical A. fumigatus isolated from healthcare settings in 37 states (13) and environmental A. fumigatus isolated from agricultural settings in Florida and Georgia (11,20).We placed our new isolates and data for 295 publicly available isolates on a map allowing visualization of the distribution of all clinical and environmental A. fumigatus so far reported in the U.S. (Fig. 1; Table S1).It is impossible to determine the actual frequency of azole-resistant A. fumigatus in individual states or associated with particular crops or substrates because sampling intensity and isolate screening and selection have varied widely; however, it is clear that azole-resistant A. fumigatus is widespread geographically and across crops and substrates.Pan-azoleresistant isolates were prevalent among samples from grape, compost, tulips, and hemp (Table 1). A. fumigatus isolates have been reported from 38 states.Of these isolates, 241 were collected from environmental settings in 9 states and 189 from clinical settings in 37 states; 263 were sensitive to clinical azole drugs, 101 to a single azole, and 66 to  more than one azole (pan-azole resistant).Including the present study, agricultural sites in only nine states have been sampled; azole-resistant A. fumigatus was not detected in agricultural samples from Virginia but was detected in agricultural samples from the other eight states surveyed (Fig. 1; Table S1).
Several studies showed that worldwide A. fumigatus populations fell into two clusters (Clade A and Clade B) with most TR 34 /L98H and TR 46 /Y121F/T289A-based pan-azole resistance in Clade A (12)(13)(14).A recent pan-genome study including more isolates from around the world showed that A. fumigatus populations fell into three clusters: Clade 1 contains fewer azole-resistant strains and appears to be the same as the previously identified Clade B; Clade 2 contains many TR-based pan-azole isolates and appears to be the same as Clade A. The small Clade 3 has no azole resistance and is made up of 14 clinical isolates from Europe, the U.S., Canada, and a single environmental isolate from Peru (16).To determine the relationships of U.S. agricultural isolates from the current study to worldwide A. fumigatus clinical and agricultural populations, we performed whole-genome sequencing on 135 and used the resulting sequences, along with 594 publicly available whole-genome sequences to construct a neighbor-joining tree of 729 total sequences (Fig. S1; Table S2).To better visualize the tree, 482 representative isolates were chosen for subsampling (Fig. 2; Table S2).In addition to analyzing azole-resist ance mutations in cyp51A, we analyzed genes responsible for resistance to agricultural fungicides [benA for benzimidazole (MBC) class, cytB for quinone outside inhibitor (QoIs), and sdhB for succinate dehydrogenase inhibitor (SDHI) class] and mating-type (MAT1-1-1, MAT1-2-1; AY898660.1,AFUA_3G06170).White stripes on pie charts denote azole-resistant isolates.It should be noted that samples in the field crop category in Georgia are primarily from a single peanut cull pile in which multiple azole-resistant isolates were detected.Based on the current study and publicly available data from previous studies as shown in Table S1 (11, 13, 14, 20, 42-50).The map was created using the Cran-R project (https://cran.r-project.org/web/packages/usmap/index.html).Our phylogenetic analysis of 729 worldwide isolates was consistent with three clades with the branch leading to the small Clade 3 showing 100% bootstrap support (Fig. 2; Fig. S1).MAT1-1 and MAT1-2 isolates were present in almost equal proportions through out the tree (354/729 and 375/729, respectively), and there was no association with clade designation (Fig. 2; Fig. S1).Based on sequence data and single mutations known to be associated with fungicide resistance phenotypes in A. fumigatus (11,53,54), we classified isolates as sensitive or resistant to the agricultural fungicides MBC, QoI, and SDHI (Table S2).We used MIC data for ITR, VOR, and POS to determine if isolates were sensitive and resistant to a single clinical azole (azole resistant) or to multiple clinical azoles (pan-azole resistant).Because MICs for all three clinical azoles were not reported for all publicly available strains, the actual number of pan-azole-resistant isolates might be higher than our data suggest.Clade 1 was the largest containing 463/729 total isolates.A majority of Clade 1 isolates were azole-sensitive (292/463) and of the 171 azole-resistant isolates, 80/171 were resistant to more than one azole, and only five had TR mutations in the cyp51A promoter.No Clade 1 isolates had the known mutations associated with resist ance to MBC, QoI, or SDHI agricultural fungicides.Clade 2 was the next largest group containing 208/729 isolates.Most Clade 2 isolates were azole-resistant (154/208) with 103 being pan-azole resistant and 128 carrying a TR mutation in the cyp51A promoter.Seventy-nine Clade 2 isolates had the known mutations associated with resistance to MBC, QoI, or SDHI agricultural fungicides with 53/79 indicating resistance to more than one agricultural fungicide (multi-fungicide resistant).Interestingly all fungicide-resistant isolates in Clade 2 also contained a TR mutation (79/79).Clade 3 was the smallest (39/729) and contained azole-sensitive (17/39), azole-resistant (20/39), pan-azoleresistant (2/39), and fungicide-resistant isolates (4/39), though no TR or multi-fungicide resistance mutations were present.
To further investigate population structure, all 729 isolates were included in a PCA.Isolates congregated in three main clusters supporting a three-clade structure with some presumed recombinant isolates intermixed among all clades (Fig. 3).
To further investigate presumed recombination between clades, we used our 480 isolate subsample group of A. fumigatus U.S. agricultural and worldwide isolates to conduct ADMIXTURE analysis.ADMIXTURE was run with five different seeds for K values 2 through 20, and the best log likelihood K values were analyzed (Fig. S2 and S3).K3 best fits the data and supports three clusters that mostly align with the three clades but also show gene flow between clades (Fig. 4).
Our phylogenetic, ADMIXTURE, and PCA analyses all suggested three populations with the small Clade 3 being the most diverged from the other two.To determine whether Clade 3 might be a cryptic species distinct from A. fumigatus, we constructed gene trees for eight different housekeeping genes (act1, efg1, gapdh, histH4, ITS, rpb2, sdh1, and tef1) from all 729 isolates with A. fischeri as an outgroup (Fig. 5; Fig. S4).In no case did Clade 3 form a monophyletic group that diverged from Clades 1 and 2 as would be expected if it was a different species nor did Clade 3 group with A. fischeri.In six of the eight trees, Clade 3 isolates were mostly clustered together but not monophyletic, and Clades 1 and 2 isolates were interspersed suggesting Clade 3 is rapidly diverging from Clades 1 and 2 (Fig. S4).In two of the eight trees, gapdh and ITS isolates from all three clades were interspersed showing less divergence among clades for these specific loci.The absence of monophyly for the gene trees for clade 3 isolates shows current or historically recent gene flow and thus indicates that it is not a distinct species.Before the current study, A. fumigatus isolates from clinical sources had been reported in 37 states and from environmental sources in two states [Georgia and Florida (11,20,55); Fig. 1].We surveyed agricultural sources in seven additional states including four on the eastern U.S. coast and three on the western U.S. coast.We combined our data with publicly available data to get an overview of A. fumigatus reported from U.S. clinical and environmental sources.Sampling intensity has varied widely, so we could not determine the true prevalence of azole-resistant A. fumigatus in each state or associated with different crops.However, it is clear that azole-resistant A. fumigatus is widespread in the U.S. We found it in agricultural environments on both coasts where a variety of crop types have been cultivated.Interestingly, we found high levels of TR-based azole resistance and multi-fungicide resistance in isolates from tulip farms on the west coast that also grow hemp (Table S1).Most tulips in the U.S. are grown from bulbs imported suggests that the genetic background of Clade 2 isolates allows more rapid adaptation to environmental stress.Though we found no TR-based resistance in Clade 3, we did find two pan-azole-resist ant isolates that did not carry a TR allele and 20 isolates that were resistant to the single-azole POS.Generally, our study found more POS resistance (and hence non-panazole resistance) than reported in other studies.We think this is because MICs are less often determined for POS than for ITC and VOR.In our cumulative data set, 35% of isolates do not have MICs reported for POS, while only 14% lack MICs for ITC and VOR (Table S2).

U.S. populations show signatures of recombination
Previous work from others found evidence of very high recombination rates in A. fumigatus with recombination frequently occurring between clades (16,60).Our isolates also showed signatures of recombination between clades in PCA and ADMIXTURE analysis (Fig. 3 and 4).

Conclusion
Our study expands environmental sampling of A. fumigatus in the U.S. We show that azole-resistant and multi-fungicide-resistant isolates are found in agricultural settings in both the east and west coast regions of the country in areas where a variety of crops have been cultivated.We show support for three clades of A. fumigatus, consistent with a recent study that included many fewer U.S. isolates (Fig. 3 and 4) (16).We found that while the small Clade 3 has no TR-based azole resistance, it contains isolates with resistance to single and multiple azoles and to the QoI fungicides.Interestingly, the remaining 79 isolates that were resistant to agricultural fungicides in the MBC, QoI, or SDHI classes were in Clade 2, and all also carried a TR allele of cyp51A associated with pan-azole resistance.U.S. isolates also showed high levels of recombination raising the worrying prospect that resistance to clinical azoles will continue to spread in the U.S.

FIG 1
FIG 1 Locations of all A. fumigatus isolated from clinical and environmental settings in the U.S. as of June 2023.States with gray background have no reported environmental sampling of A. fumigatus.The size of the pie charts represents the number of isolates reported in each state.Sampling intensity varied widely, so the isolate number does not accurately reflect the relative abundance of A. fumigatus in each state.Pie chart colors represent sample types (gray: soil amendments; yellow: field crops; purple: flowers and herbs; green: annual fruits and vegetables; red: perennial fruits and vegetables; dark blue: human clinical).

Full 8 FIG 2
FIG 2Neighbor-joining tree of 480 subsampled environmental and clinical isolates of A. fumigatus.Whole-genome sequences from agricultural sites on the east and west coasts of the United States (eAF1XXX) were analyzed along with publicly available data (TableS1).Af293 was used as the reference genome and root of the tree.Bootstrap values greater than 95% are indicated by a dot on the branch.Clade 1 branches are black; Clade 2 branches are red; Clade 3 branches are blue.Country of origin is listed next to each isolate according to their two-letter designation (CA, Canada; DE, Germany; ES, Spain; GB, Great Britain; IN, India; NL, Netherlands; US, United States).North American isolates are shown with a green background, European with gold, and Asian with blue.Green and blue bars indicate environmental and clinical isolates, respectively.Black and white bars represent mating types MAT1-1 and MAT1-2, respectively.Open red circles indicate azole-resistant isolates.

Full
A. fumigatus is widespread in the U.S.

FIG 3
FIG 3 Principal components (PC) 1 and 2 plot mostly separate the three clades.Clade 1 is represented by black dots.Clade 2 is represented by red dots.Clade 3 is represented by blue dots.PC 1 represents 41.59% of data along the x-axis.PC 2 represents 13.04% of data on the y-axis.

FIG 5
FIG 5 Gene trees of housekeeping genes show that Clade 3 is not monophyletic.Gene trees of act1 (shown) and seven other housekeeping genes (Fig. S4) from 729 A. fumigatus isolates and A. fischeri as outgroup show that Clade 3 is not monophyletic, supporting the view that these isolates are A. fumigatus and not a cryptic species.Circles on branches represent bootstraps over 90.Black squares represent isolates from Clade 1. Red squares represent isolates from Clade 2. Blue squares represent isolates from Clade 3.

TABLE 1
Sampling of A. fumigatus strains from agricultural sites

TABLE 1
Sampling of A. fumigatus strains from agricultural sites (Continued)

sampled Sampling date(s) MM/DD/YY (no. sites) Isolates collected, no. Whole-genome sequencing strains (*azole-resistant) b Total # isolates obtained (# tested) MIC S Resistant
a # tested indicates isolates for which MIC was determined.Some isolates were resistant to more than one azole.For more detail see TableS2.b Asterisks indicate azole-resistant isolates based on minimum inhibitory concentration (MIC).Bold numbers indicate isolates resistant to more than one azole based on MIC (pan-azole-resistant isolates).c These isolates come from a field cultivated with both hemp and tulips.Full-Length Text Applied and Environmental Microbiology April 2024 Volume 90 Issue 4 10.1128/aem.01782-237